PMC:514616 / 1107-7281 JSONTXT

{"project":"2_test","denotations":[{"id":"15296510-11252569-133587950","span":{"begin":699,"end":700},"obj":"11252569"},{"id":"15296510-9039911-133587951","span":{"begin":826,"end":827},"obj":"9039911"},{"id":"15296510-9690471-133587951","span":{"begin":826,"end":827},"obj":"9690471"},{"id":"15296510-10331804-133587951","span":{"begin":826,"end":827},"obj":"10331804"},{"id":"15296510-10993078-133587951","span":{"begin":826,"end":827},"obj":"10993078"},{"id":"15296510-10947985-133587951","span":{"begin":826,"end":827},"obj":"10947985"},{"id":"15296510-8598906-133587951","span":{"begin":826,"end":827},"obj":"8598906"},{"id":"15296510-8598906-133587952","span":{"begin":991,"end":992},"obj":"8598906"},{"id":"15296510-15117786-133587953","span":{"begin":1083,"end":1084},"obj":"15117786"},{"id":"15296510-9190674-133587954","span":{"begin":1219,"end":1221},"obj":"9190674"},{"id":"15296510-9057968-133587955","span":{"begin":1222,"end":1224},"obj":"9057968"},{"id":"15296510-5216343-133587956","span":{"begin":1804,"end":1806},"obj":"5216343"},{"id":"15296510-6790319-133587956","span":{"begin":1804,"end":1806},"obj":"6790319"},{"id":"15296510-1811135-133587956","span":{"begin":1804,"end":1806},"obj":"1811135"},{"id":"15296510-1725801-133587956","span":{"begin":1804,"end":1806},"obj":"1725801"},{"id":"15296510-16589125-133587957","span":{"begin":1884,"end":1886},"obj":"16589125"},{"id":"15296510-13912738-133587958","span":{"begin":2419,"end":2421},"obj":"13912738"},{"id":"15296510-8898230-133587959","span":{"begin":2581,"end":2583},"obj":"8898230"},{"id":"15296510-12050139-133587960","span":{"begin":3293,"end":3295},"obj":"12050139"},{"id":"15296510-11223880-133587961","span":{"begin":4337,"end":4339},"obj":"11223880"},{"id":"15296510-11754016-133587961","span":{"begin":4337,"end":4339},"obj":"11754016"},{"id":"15296510-4635463-133587961","span":{"begin":4337,"end":4339},"obj":"4635463"},{"id":"15296510-11223880-133587962","span":{"begin":4405,"end":4407},"obj":"11223880"},{"id":"15296510-12592573-133587963","span":{"begin":4408,"end":4410},"obj":"12592573"},{"id":"15296510-6894203-133587964","span":{"begin":4954,"end":4956},"obj":"6894203"},{"id":"15296510-17830958-133587965","span":{"begin":5151,"end":5153},"obj":"17830958"},{"id":"15296510-14748925-133587966","span":{"begin":5591,"end":5593},"obj":"14748925"}],"text":"Background\nThe study of reproduction and its artificial manipulation is important in many fields. For example, in sea urchins, an animal's testes can be dissected and sperm is activated by exposure to seawater. Eggs can be released by injecting KCl into the perivisceral cavity, and mixing eggs and sperm in vitro produces fertilization, as evidenced by the appearance of the fertilization membrane and subsequent development of embryos [1]. These simple techniques have been the basis for such dissimilar studies as those of Berdishev [1], dealing with the role of fatty acids and cannabinoids in fertilization, to investigation of the gene expression patterns of hybrids by Nielsen and coworkers [2].\nArtificial reproduction has also been well-studied in mammals, and cloning of eutherians from somatic cells is now common [3-8]. Harvested eggs can be enucleated and merged with a somatic cell and the reconstructed embryos cultured in vitro before being implanted into surrogate mothers [8]. These methods have opened up new possibilities in both basic and applied science [e.g. [9]]. Importantly, artificial fertilization has been utilized as a means of assisting with the conservation effort of declining species [10,11].\nFrogs have been favorite model organisms in reproductive and developmental biology for many years, mainly because of the ease with which they can be kept in captivity; their external fertilization; easily visible development in large, transparent eggs; and large numbers and ease of manipulation of their eggs. Consequently, research on frogs has often been in the vanguard of advancement in artificial reproduction techniques, and much is known about a few model species such as the African clawed frog, Xenopus laevis and the North American leopard frog, Rana pipiens [e.g. [12-15]]. Indeed, the first vertebrate cloned from a somatic nucleus was a frog [16]. Briggs and King injected female R. pipiens with male pituitary glands to induce ovulation and deposition of unfertilized eggs. The eggs were mechanically activated by pricking with a needle, a process which brings the pronucleus immediately under the surface of the animal pole. Taking advantage of this situation, the pronuclei were extruded, along with a small amount of cytoplasm, using a glass needle. In other species, such as the Xenopus or the axolotl, UV radiation can be used to destroy the female pronucleus instead [17,18]. Development was then directed by a somatic nucleus microinjected into the cytoplasm of the enucleated egg.\nIn another group of experiments, Kroll and Amaya [19] developed an effective and reliable method for creating transgenic Xenopus: testes were macerated in solution and the sperm membranes partially dissolved, allowing access to the condensed chromosomes. Linearized bacterial plasmids containing genes of interest were mixed in with the sperm solution and recombinant ligase was used to covalently insert the bacterial plasmids into the sperm genomic DNA, resulting in the insertion of many copies of the plasmid construct into each genome. These nuclei were then microinjected into mature eggs, generating, under appropriate conditions, hundreds of nonmosaic, transgenic embryos. Such techniques allow the investigation of gene function in these species [e.g. [20]].\nClearly, there are enormous advantages to being able to manipulate a species' reproduction in the laboratory. However, despite the multiplicity of studies concentrating on anurans, to date all model species are aquatic breeders. Yet amphibians have the largest diversity of breeding strategies among terrestrial vertebrates, and it is to be expected that species with different reproductive strategies will require different methods for their manipulation in the laboratory. Therefore, many species remain experimentally intractable. Notably, terrestrial-breeding frogs, a very large and diverse group of organisms, are largely inaccessible to reproductive investigations.\nThe neotropical frog genus Eleutherodactylus is characterized by terrestrial breeding and direct development without an aquatic larval stage. With more than seven hundred described species, this is the largest vertebrate genus [21,22]. There has been considerable experimental attention focused on Eleutherodactylus frogs, ranging from basic developmental biology [23-26]; to ecology [e.g. [27-30]]; to the evolution of development [23,31]. However, there are as yet no available techniques for performing in vitro fertilization in these frogs. The development of such techniques would allow additional investigations into the genetic regulation of direct development in these species and would also assist with conservation of declining populations, an important goal considering the fact that many species of Eleutherodactylus are declining, and several are already extinct [32,33].\nEleutherodactylus coqui are small tree frogs with internal fertilization and direct development [34]. This species is extremely common in the forests of Puerto Rico, and it has been found that their population size is limited by the availability of retreat sites, as opposed to food resources [35]. As with all other studied Eleutherodactylus species, E. coqui embryos develop directly into tiny froglets in terrestrial eggs, without a tadpole stage [36]. Protocols for the husbandry of these frogs have been reported, and it is possible to maintain them in the laboratory for multiple generations [37,38]. A method has also been developed to induce ovulation using an artificial form of luteinizing hormone-releasing hormone (LHRH) [39]. It is know in this species that sperm entry occurs at a small disc at the animal pole of the egg, and that polyspermy is apparently common but does not interfere with development [40]. Cortical granules and their exocitosis have also been described using electron microscopy [40], but the large (5 mm diameter), opaque and featureless eggs make it difficult to observe the rising of a fertilization membrane. As E. coqui is arguably the best-studied terrestrial-breeding frog, we have focused on this particular species as a model for the development of reproductive techniques."}